All the primers used had
been reported and optimized in previous studies [34,35]. The
transcription of CD4 and CD8 Th cell markers was also assessed
using the primers CCTGCTCATCCACAGCCTAT (F) and
CTTCTCCTGGCTGTCTGACC (R) for CD4 and AGTCGTGCAAAGTGGGAAAG
(F) and GGTTGCAATGGCATACAGTC (R) for CD8. The corresponding
accession numbers are AY973030.1 and NM_001124263,
respectively. All qPCR reactions were performed in triplicate and for
each mRNA, and gene expression was normalized to that of the
endogenous control (elongation factor 1-a; EF1-a) and expressed as
2DCt, where DCt was determined by subtracting the average EF1-a
Ct value from the average target Ct. The change in expression
relative to the empty plasmid pcDNAwas also determined for some
of the samples by applying the formula 2DDCt [45] where
DDCt ¼ DCt of samples of target gene DCt of the calibrator (pcDNA
control).